Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors that are differentially expressed by a number of immune and non-immune cell populations. Certain CRs have been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. CCR5 and CXCR4 have been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 and CXCR4 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) or oxidation of cholesterol significantly reduced the binding and signaling of CXCL12 and CCL4 using CXCR4- or CCR5-expressing T cells, respectively. Antibodies specific for distinct CXCR4 or CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction and cholesterol oxidation. These results suggest that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of chemokine receptors. Lipid rafts also play an important role in signal integration and cellular activation of a number of cytokine and growth factor receptors. Flotillin proteins have recently been shown to be recruited to lipid raft microdomains upon cellular activation and have been implicated in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins in T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin-1 but not flotillin-2 redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12-treated T cells, flotillin-1 also associates with several raft proteins including LAT, Lck, CD48 and CD11a. In addition, an increase in CXCR4 association with flotillin-1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin-1 resulting in an inhibition of CXCL12-mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the association of flotillin-1 with lipid raft during chemokine exposure may play an important role in chemokine receptor recruitment to and signaling in lipid rafts and possibly in leading edge formation. Overall, we believe that a greater understanding of the various signaling and cell surface proteins associated with lipid rafts may provide great insight into age-related alterations in cell signaling and migration.